Immunohistochemical Expression Patterns of S100, Synapto

JBUON 2018; 23(5): 1540-1545 ISSN: 1107-0625, online ISSN: 2241-6293 • www.jbuon.com E-mail: [email protected]

ORIGINAL ARTICLE Immunohistochemical expression patterns of S100, synaptophysin, chromogranin A and neuron specific enolase in predicting malignant behaviour in paragangliomas Constantinos Nastos1, Anneza Yiallourou2, Thomas Kotsis1, Maria Mizamtsidi3, Dionysios Delaportas1, Agathi Kondi- Pafiti4, George Polymeneas1 12nd Department of Surgery, School of Medicine, Aretaieion Hospital, National and Kapodistrian University of Athens, Greece; 2Surgical Division, Medical School, University of Cyprus, Nicosia, Cyprus; 3Endocrinology Department, Hellenic Red Cross, Korgialeneio-Benakeio Hospital, Athens, Greece; 4Pathology Department, School of Medicine, Aretaieion Hospital, National and Kapodistrian University of Athens, Athens, Greece

Summary

Purpose: The purpose of this study was to evaluate the role ratio was 1.75: 1. The mean age was 43.5 and 51.6 years for of immunohistochemical markers in the prediction of malig- women and men, respectively. In 5 patients the tumors had nancy in paragangliomas. malignant clinical behavior. Their mean size was 3.65 cm for benign and 4.56 cm for malignant neoplasms. NSE expres- Methods: Our institute’s patient records between 1990-2012 sion was diffuse in 47.1% and 0% for benign and malignant were retrieved in order to identify patients who were treated tumors, respectively (p=0.10). S100 expression in the periph- for paragangliomas. Size and location of the tumour, exist- ery of the tumour was typical in 88.2% and 0% for benign ence of concurrent metastatic disease, patient demograph- and malignant tumors, respectively (p<0.001). ics and survival were recorded. Haematoxylin-eosin stained slides were reviewed and all tumours were stained specifically Conclusion: Immunohistochemical profile from the com- for neuron specific enolase (NSE), chromogranin, synapto- bination of NSE, synaptophysin chromogranin and S100 physin and S100 protein positivity. Positivity and expression staining patterns can serve as a cheap and valuable tool patterns of the above markers were evaluated and compared for correctly distinguishing between malignant and benign between malignant and benign tumours. Malignant behav- paragangliomas with high diagnostic accuracy. iour was defined when patient had concurrent or subsequent lymph node involvement, local recurrence and/or metastases. Key words: chromogranin, immunohistochemistry, ma- Results: A total of 22 patients with a diagnosis of para- lignant paragangliomas, neuron-specific enolase, S100, ganglioma were treated in our institutes. Female to male synaptophysin

Introduction Paragangliomas represent rare tumours aris- in the neck and base of the skull, mostly deriving ing from sympathetic or parasympathetic neuronal from the carotid body. On the contrary, paragangli- tissue and are chromaffin cell tumours. They devel- omas originating from ganglia of the sympathetic op from neural crest cells and comprise of 10-20% nervous system can be located along the distribu- of all chromaffin cell tumours [1]. tion of the sympathetic nervous system from the Paragangliomas arising from ganglia of the head and neck, along the aorta through the me- parasympathetic system are almost always located diastinum and in the retroperitoneal space, up to

Correspondence to: Constantinos Nastos, MD, PhD. Second Department of Surgery, School of Medicine, National and Kapodis- trian University of Athens, Aretaieion University Hospital, 76 Vassilisis Sofias Ave, 11528 Athens, Greece. Tel: +30 210 7286000, Fax: +30 210 7286128, E-mail: [email protected] Received: 24/01/2018; Accepted: 17/02/2018 Combination of tumor markers predicting malignant paragangliomas 1541 the prostate gland and the urinary bladder. Most on a Ventana Automatic System (Benchmark XT) with of them are located in the abdomen (75%), mostly the use of iVIEW DAB Detection kit - an indirect biotin- at the site of the origin of the inferior mesenteric streptavidin system using DAB as chromogen. Protocols artery where the Zuckerkandl organ is located. and primary antibodies used for specific staining of NSE, The majority of sympathetic system paragan- chromogranin A, S100 protein and synaptophysin are described below in more detail. gliomas secrete catecholamines, which can be a For NSE we used a mouse monoclonal antibody life-threatening condition, and are associated with (clone: BBS/NC/VI-HI4- Dako, Denmark), with a dilu- hormone secretion syndromes. On the other hand, tion of 1:400, followed by 8-min of epitope retrieval and parasympathetic paragangliomas do not often se- 16-min incubation of the antibodies. crete catecholamines. For chromogranin A staining we used a mouse Paragangliomas may have malignant behav- monoclonal antibody (clone: LK2H10- Biocare Medical, iour, with a prevalence of 2-26% [2]. The most com- Concord, CA, USA), with a dilution of 1:50, followed by mon metastatic sites are lymph nodes, bone and 30-min of epitope retrieval and 20-min of incubation of lung. Although benign paragangliomas have a very the antibody. good 5-year overall survival of more than 90%, For synaptophysin, a rabbit monoclonal antibody paragangliomas with malignant behaviour have (clone: SP11- Thermo Scientific, Fremont CA, USA) with dilution of 1:80 was used, followed by 30-min of epitope 5-year survival of around 50% [3,4]. It is therefore retrieval and 28-min incubation of the primary antibody. imperative that these tumours should be recog- Finally, for S-100 protein staining we used a mouse nized early so that more aggressive surgical and monoclonal antibody (clone 4C4.9- Cell Marque Rock- medical treatment can be adopted. lin, CA, USA) with a dilution of 1:100, followed by 30- Although many attempts have been made to min of epitope retrieval and 8-min of primary antibody identify clinical, biochemical and pathological incubation. markers suggestive of malignancy, none of them seem to be quite sensitive in predicting malignan- Staining pattern interpretation protocol cy, leading to undertreatment of these patients. All cases were reviewed by a single expert patholo- Immunohistochemical markers have shown insuf- gist (A.P). Immunohistochemical staining patterns were ficient concordance. Therefore, attempts are ad- characterized according to Table 1. Each individual stain dressed towards designing multi-parameter scor- pattern was scored and the total score was calculated and ing systems for risk stratification. evaluated for being predictive of malignancy. The purpose of this study was to retrospec- Statistics tively evaluate the expression and diagnostic accuracy of immunohistochemical markers and their Results were expressed as mean ± SD. Comparison between groups was performed using the x2 test for combination for the prediction of malignancy of qualitative data and the unpaired t-test for quantitative paragangliomas. data. Sensitivity (true positive ⁄ true positive + false nega- Methods tive), and specificity (true negative ⁄ true negative + false positive) were calculated. Statistical analyses were per- Patient records between 1990-2012 were retrieved in order to identify those treated in our departments with a diagnosis of paraganglioma. Table 1. Scoring of immunohistochemical staining pat- Patient gender, age, size and location of the tumor, terns the presence of lymph node involvement or metastatic disease and the final pathology report were recorded. Immunohistochemical stain Staining pattern Score Follow-up data were collected either from the oncol- Neuron specific enolase Sporadic cells 1 ogy service department of our institutes or after direct Foci of cells 2 communication with the patients, when data were not Diffuse stain 3 available. Metastatic behavior was determined based on the Synaptophysin Sporadic cells 1 evaluation of lymph node involvement, distant organ Foci of cells 2 involvement and recurrence of disease. Diffuse stain 3 The study protocol was approved by the hospitals’ S100 No positivity 1 Ethics Committees. Sporadic cells 2 Immunohistochemistry protocol Peripheral staining of 3 tumors Tissue samples were fixed in 10% buffered formalin Chromogranin A Sporadic cells 1 and were embedded in paraffin. Sections of 5 μm were stained with hematoxylin-eosin. Immunohistochemis- Foci of cells 2 try was performed on deparaffinized 3-5 μm sections Diffuse stain 3

JBUON 2018; 23(5): 1541 1542 Combination of tumor markers predicting malignant paragangliomas formed using a commercially available statistical soft- ware package (SPSS 17.0 for Windows; SPSS Inc., Chi- cago, IL, USA). A p value less than 0.05 was considered statistically significant.

Results

Twenty-two patients were found with tumors classified as paragangliomas. Patient records, im- aging studies, pathology workup and follow up were identified in 17 patients with benign disease and in 5 patients with paragangliomas with malignant behavior either at first presentation or during patient follow up. Fourteen patients were female and 8 male, with a mean age of 45.2±14.2 and 50.6±19.4 years, respectively. Mean size of the Figure 1. Histological section of a paraganglioma show- neoplasms was 3.6±1.5 cm for benign and 4.5±2.7 ing a positive S100 immunoreaction of the sustenctacular cells surrounding the neoplastic nests (immunostain S100 cm for malignant tumors (p=0.34). Data are sum- ×120). This is a case of a non malignant paraganglioma. marized in Table 2. NSE expression pattern was characteristic with foci of cells stained in both malignant and benign tumors. There was a trend towards more diffuse staining pattern in benign tumors, that, however, did not achieve statistical significance (p=0.10). The expression pattern of S100 was significantly different between groups with staining at the periphery of the tumors in the majority of benign tumors (p<0.001). This typical staining pattern was absent in all malignant tumours (Figures 1 and 2). Moreover, there was a significant difference in the expression of synaptophysin between benign and malignant tumors, with malignant neoplasms showing a more sporadic expression in contrast to benign tumors in which the stain was organized in cell foci or diffusely (p=0.003). No difference in the Figure 2. Histological section of a paraganglioma show- expression pattern of chromogranin A was identi- ing random positive S-100 immunoreaction of the sus- fied between the two groups (Figure 3). Data are tenctacular cells (immunostain S100×120). This is a case summarized in Tables 3 and 4. of a metastatic paraganglioma.

Table 2. Demographic data and tumor characteristics (mean±SD)

Characteristics Age (years) Cervival location Retroperitoneal location Tumor size (cm) Male/ Female

Benign 45.2±14.2 13 4 3.6±1.5 6/11 Malignant 50.6± 19.4 2 3 4.5±2.7 2/3 p 0.50 1.24 1.24 0.34 0.84

Table 3. Differences in immunohistochemical staining patterns of neuron-specific enolase and S-100

Tumour Neuron specific enolase S100

Sporadic cells Foci of cells Diffuse stain Negative stain Sporadic cells Peripheral staining

Benign 1 8 8 0 2 15 Malignant 0 5 0 4 1 0 p 0.10 <0.001

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Table 4. Differences in immunohistochemical staining patterns of synaptophysin and chromogranin

Tumour Synaptophysin Chromogranin

Sporadic cells Foci of cells Diffuse stain Sporadic cells Foci of cells Diffuse stain Benign 0 10 7 0 10 7 Malignant 3 1 1 0 4 1 p 0.03 0.61

There was a significant difference in the total score of the immunohistochemical staining pattern between patients with benign and malignant disease (p<0.001). When using a cut-off value of 8 for the total immunohistochemical staining score, all patients with total scoring less than 8 had a tumor of malignant behavior, as opposed to patients with a score of 9 or higher whose tumor demonstrated a benign clinical course during follow-up. The absence of peripheral and typical S100 staining has a sensitivity of 100% and a specificity of 88.2% in predicting malignancy in our series. A total immunohistochemical pattern score less than 8 had a specificity and sensitivity of 100 in predicting malignancy, as shown in Table 5 and Figure 4. Figure 3. Histological section of a paraganglioma showing positive chromogranin immunoreactions of the cells (immunostain ×120). Discussion

Paragangliomas are chromaffin cell tumors that develop from the neural crest cells. They are divided into tumors of the parasympathetic or sympathetic ganglia. The parasympathetic paragangliomas are found mainly in the neck and skull base and arise within the carotid body or the globus jugulotympanic. Parasympathetic paragangliomas are the most common type, while the rest are de- rived from the sympathetic ganglia and are located in the chest and retroperitoneal space and rarely to the head and neck region [5]. Both sympathetic and parasympathetic paragangliomas have similar histological characteristics, although there are data supporting that they are genetically different [6-8]. Figure 4. Histological section of a typical paraganglioma The majority of paragangliomas are benign. (Hematoxylin-Eosin ×120). However, malignancy is thought to exist in up to 35% of sympathetic and, rarely, of parasympathetic (head and neck) paragangliomas. These figures can Table 5. Differences in total immunohistochemical stain- be even higher when specific genetic syndromes, ing pattern score between benign and malignant paragangliomas such as succinate dehydrogenase gene mutations (SDHB, SDHC, SDHD), neurofibromatosis type 1 Tumour Total immunohistochemical pattern score (NF-1), von Hippel-Lindau, the Carney triad, and others are taken into account [4,8]. Score ≤8 Score ≥ 9 To date there is no established clinical, bio- Benign 0 17 chemical, radiological or pathological features of Malignant 5 0 malignancy for these tumors. According to cyto- morphometric studies, predictors of malignancy in p <0.0001 a paraganglioma are considered to be extra-adrenal

JBUON 2018; 23(5): 1543 1544 Combination of tumor markers predicting malignant paragangliomas location, confluent tumor necrosis, vascular inva- from adjacent tissue or the circulation [14]. S100 sion, local invasion, coarse nodularity and absence staining in type II cells is localized at the periph- of hyaline globules. The diagnosis of malignancy ery of the cytoplasm and of the nuclei. Abundance is made when regional lymph node metastasis or of type II cells may offer good prognosis of para- distant metastases are found either at diagnosis or gangliomas [15]. Interestingly, in the majority of during follow-up of these patients. Although there published case series, a remarkable decrease in have been many attempts to correlate histological the immunoreactivity of S100 protein in malig- features with malignant potential of these tumors, nant cases has been noted. S100 positive cells were all of them lack specificity and sensitivity. Multipa- noted in metastatic deposits, suggesting that the rameter scoring systems have been developed for sustentacular cells had metastatic potential along risk stratification, such as the Phaechromocytoma with chromaffin cells and are an integral part of the of the Adrenal Scaled Score (PASS) [9] and the Grad- tumour [9]. In our study, the expression pattern of ing System for Adrenal Phaechromocytoma amd S100 was significantly different between groups Paraganglioma (GAPP) [10]. Although the features with a staining of the periphery of the tumors in integrated in these systems may have some valid- the majority of benign tumors (p<0.001). ity, PASS was found to have very poor concord- Locally aggressive tumor tissues express poor- ance between expert pathologists in a validation ly chromogranin and S100 protein [16]. Synapto- study [11], and GAPP has so far had no validation physin expression is not limited by the stage of study results published. Malignant characteriza- differentiation, as its monoclonal variant is very tion remains of vital importance, as several man- sensitive and specific for neuroendocrine tumors. agement options have been made available, from In our patients’ histochemical analysis, there was 131I-MIBG treatment (and recently peptide recep- a significant difference in the expression of synap- tor radionuclide treatment), to chemotherapy and tophysin between benign and malignant tumors, external beam radiation therapy, even in the adju- with malignant neoplasms showing a more spo- vant setting after surgery [12]. In addition, predic- radic expression in contrast to benign tumors in tion of malignancy in these tumors could lead to a which the stain was organized in cell foci or dif- more aggressive treatment which may potentially fusely (p=0.003). increase survival rates. Pathologists consider that NSE is a sensitive Several immunohistochemical markers have marker for neuroendocrine tumors, although it has been widely incorporated in the pathology workup been reported to have a low specificity. Regarding of neuroendocrine neoplasia, such as enzymes neuroendocrine tumors’ malignant behavior, previ- (NSE), proteins stored in the secretory granules ous studies have claimed that NSE may reflect cell (chromogranin A and HISL- 19 protein), resident necrosis, and raised NSE levels are associated with proteins of the presynaptic vesicles, catechola- poorly differentiated tumors. mines and indolamines, neuropeptides, and mol- ecules with unknown function (Myelin associated Conclusion glycoprotein Leu- 7). Chromogranins, in general, are considered to Distinguishing between benign and malignant provide sufficient documentation of neuroendo- paragangliomas remains a diagnostic challenge for crine differentiation. Chromogranin A, in particu- every pathologist, due to the absence of established lar, has been found to reflect both tumor burden criteria. Combining immunohistochemical staining and tumor secretion in neuroendocrine tumors [13]. patterns of routine markers can be proved to be S100 protein is used to identify sustentacular a cheap, readily available and reliable method of cells. In normal paraganglia, these cells exhibit predicting malignancy in these tumours. various functions, such as serving as stem cells, and as glial-like supporting cells. In paraganglio- Conflict of interests mas, however, their role is questioned, because they could be either neoplastic or benign migrants The authors declare no conflict of interests.

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